Confocal laser scanning microscopy (CLSM) is a valuable tool for obtaining high resolution images and 3D reconstructions by using a spatial pinhole to eliminate out-of-focus light or flare. This technology permits one to obtain images of various Z-axis planes (Z-stacks) of the sample. The detected light from an illuminated volume element within the specimen represents one pixel in the resulting image. As the laser scans over the plane of interest, where the beam is scanned across the sample in the horizontal plane by using one or more (servo-controlled) oscillating mirrors, the whole image is obtained pixel by pixel and line by line. Information can be collected from different focal planes by raising or lowering the microscope stage. Then the computer can calculate and generate a three-dimensional volume data of the specimen by assembling a stack of these two-dimensional images from successive focal planes.
However, the 3D image has a lower resolution in the Z-axis direction (e.g., about 1.2 μm/slice) than in the X-axis and Y-axis directions (about 0.15 μm/pixel) under the limitation of mechanical or physical properties. And the lower resolution in the Z-axis direction hampers the spatial reliability of the reconstructed high resolution neural network images, especially when comparison of two different samples is necessary. One of the inventors, Ann-Shyn Chiang, has disclosed an aqueous tissue clearing solution in U.S. Pat. No. 6,472,216 B1. In the '216 patent, the depth of observation may reach the level of hundreds micrometers. In the currently developing method, fluorescent molecules are attached to or combined with the biological tissue. Thus, making the tissue become transparent is a break-through for the observation in depth. And the way of solving the bottleneck of the Z-axis resolution is greatly needed.